• Parasites, Hosts and Diseases
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• 제41권4호
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• pp.189-196
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• 2003

In order to evaluate the possible roles of secretory proteases in the pathogenesis of amoebic keratitis, we purified and characterized a serine protease secreted by Acanthamoeba lugdunensis KA/E2, isolated from a Korean keratitis patient The ammonium sulfate-precipitated culture supernatant of the isolate was purified by sequential chromatography on CM-Sepharose, Sephacryl S-200, and mono Q-anion exchange column. The purified 33 kDa protease had a pH optimum of 8.5 and a temperature optimum of $55^{\circ}C$. Phenylmethylsulfonylfluoride and 4-(2-Aminoethyl)-benzenesulfonyl-fluoride, both serine protease specific inhibitors, inhibited almost completely the activity of the 33 kDa protease whereas other classes of inhibitors did not affect its activity. The 33 kDa enzyme degraded various extracellular matrix proteins and serum proteins. Our results strongly suggest that the 33 kDa serine protease secreted from this keratopathogenic Acanthamoeba play important roles in the pathogenesis of amoebic keratitis, such as in corneal tissue invasion, immune evasion and nutrient uptake.

• Applied Biological Chemistry
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• 제41권1호
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• pp.6-12
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• 1998

폐단백질을 활용하는 방도의 하나로 폐단백질 자원으로 부터 불용성 단백질의 분리 효율성을 높이고 기능성을 개선하기 위하여 protease를 생산하는 Aspergillus sp. MS-18 균주를 토양으로 부터 분리하고 이 균주가 생산하는 효소를 정제하여 특성을 살펴보았다. 효소 생산을 위한 최적 배양조건은 3% arabinose, 0.5% polypepton, 0.1% ammonium sulfate, 0.1% magnesium chloride 첨가로 3 일 배양이었다. 효소는 ion exchange chromatography, gel filtration 등으로 16.9 배 정제할 수 있었으며 비활성역가는 340.4 unit/mg이었다. 정제효소는 polyacryl amide gel 전기영동상 단일 밴드로 나타났으며, 분자량은 30,000 정도로 추정되었고 결정구조는 모서리가 둥그스럼한 막대 모양이었다. 정제 효소의 최적작용 pH와 온도는 9.0, $60^{\circ}C$였으며, pH 7.0-12.0까지 $50^{\circ}C$에서 안정하였다. 금속이온중 $Na^+$, $Mg^{2+}$, $Mn^{2+}$등에 의해 활성이 증대 되었으나, $Hg^{2+}$, $Cu^{2+}$, $Zn^{2+}$, $Pb^{2+}$에 의해 효소 활성이 저해되었고 저해제중 ethylenediaminetetra acetic acid와 phenyl methanesulfonyl fluoride에 의한 활성 저해가 관찰되어 금속 이온이 효소 활성에 관여하는 serine protease로 추정되었으며 정제효소의 Km, Vmax는 $29.33\;{\mu}mole/L$, $5.13\;{\mu}g/min$이었다.

• 한국미생물·생명공학회지
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• 제5권4호
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• pp.177-184
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• 1977

Arylsulfatase는 간단한 Phenols류의 황에스테르 화합물로부터 S $O_{4}$$^{[-992]}$ - 이온의 유리를 촉매한다. 이 효소는 토양센균을 포함한 많은 미생물과 동식물의 조직등에 널리 분포하여 있으며 이와 같은 넓은 분포는 이 효소의 기본적 기능이 환경학적으로 매우 중요한 의미를 갖는다고 하겠다. Arylsulfatase에 대한보고는 Klebsiella sp를 사용하여 몇몇 보고가 있다. 본 연구는 6종의 Serratia sp를 사용하여 arylsulfatase 합성조건을 검토하고 효소의 정제조건과 성질에 대하여 조사하여 Serratia marcescens를 선정하였다. Serratia mrcescens는 탄소원으로서 xylose rhamnose, glucosamine 그리고 arabinose등과 같은 몇몇 당을 이용하지 못했으며 glucose와 mannitol을 잘 이용하였으나 glucose methioniue의 경우 효소 합성을 억제시키었다. 유황원으로서는 무기유황염과 methionine의 첨가는 억제되었으며 tyramine의 첨가에 의해서 효소 함성의 억제효과는 해제되었다. 효소의 정제는 황산암모늄 포화용액의 분획과 DEAE-Cellulose, CM-Cellulose 그리고 DEAE-Sephadex A-25로 연결되는 구분 분획에 의해서 행하여졌다. 효소의 분자량은 SDS-gelelectrophoresis와 Sephadex G-100 column chromatography에 의하여 각각 46,000과 49,000으로 측정되었고 최적 PH는 6.8이었다. P-Nitrophenyl sulfate를 사용한 Km과 Vmak치는 각각 2.5$\times$$10^{4-}$M과 20 nmoles/min/mg protein이었다. 기질에 대한 특성은 phenylsulfte와 ο-, p-nit-rophenyl sulfate 그리고 p-nitro catechol sulfate에 대해서 높은 활성을 보였다. Hydroxylamine, inorganic fluoride, sulfide 그리고 Phosphate등은 강한 효소 저해작용을 나타내었고 무기유산염은 저해작용을 보여주지 않았다. Tyramine, octopamine그리고 dopamine과 같은 amino acid 또한 강한 저해 작용을 보였다.

• 생명과학회지
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• 제23권12호
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• pp.1486-1494
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• 2013

Octopus vulgaris의 장관으로부터 단백질 가수분해력과 활성을 측정함으로서 높은 단백질분해효소 생성능을 가진 균을 분리하여 동정하였다. 균이 생성한 단백질분해효소는 황산암모늄침전, cellulose CM-52 양이온 교환 크로마토그래피, DEAE-Sephadex A50 음이온 교환 크로마토그래피의 3단계를 통해 정제하였다. 장관으로부터 분리한 균중 가장 높은 단백질분해효소 생성능을 가진 균은 Bacillus sp. QDV-3로 나타났으며 이균을 분리한 후 표현형 분석, 생화학적 특성, 16S rRNA 유전자염기서열분석을 통해 Bacteria역, Firmicutes문, Bacilli강, Bacillales목, Bacillaceae과, Bacillus속으로 Bacillus flexus와 99.2%의 유사성을 보이는 것으로 확인하였다. 균이 생성한 단백질 분해효소를 QDV-E로 지정하였으며 61.6 kDa의 분자량을 나타내었다. 이 효소는 pH 9.0~9.5에서 활성을 나타내었고 최적온도는 $40^{\circ}C$였으며 $50^{\circ}C$에서는 60분간 96% 이상의 활성을 보유하였다. Phenyl methyl sulfonyl fluoride (PMSF)에 의하여 활성이 억제 되었으므로 세린 알칼리성 단백 분해 효소인 것으로 결론지었다. 금속이온인 $Mn^{2+}$ 와 $Mg^{2+}$에 의하여 효소활성 상승효과를 보였으며 $Ba^{2+}$, $Zn^{2+}$, 그리고 $Cu^{2+}$에 의하여 활성이 억제되었다.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2018년도 추계학술대회
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• pp.109-109
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• 2018

Bacillus subtilis B22, a chemotrophic and aerobic bacterial strain was isolated from homemade kimchi, identified by 16S rRNA gene sequencing. B22 was primarily screened by biochemical, carbon source utilization tests. B22 was used to produce pectinase and ${\beta}$-glucosidase by submerged fermentation under different light sources. B22 was incubated in pectin media and basal media (pH 7.0) under blue, green, red and white light-emitting diodes (LEDs), fluorescent white light, and in darkness at $37^{\circ}C$, orbital shaker 150 rpm for 24 hours. Fermentation under blue LEDs maximized pectinase production ($71.59{\pm}1.6U/mL$ at 24 h) and ${\beta}$-glucosidase production ($56.31{\pm}1.6U/mL$ at 24 h). Further, the production of enzyme increased to pectinase ($156{\pm}1.28U/mL$) and ${\beta}$-glucosidase ($172{\pm}1.28U/mL$) with 3% glucose as a carbon source. Activity and stability of the partially purified enzymes were higher at pH 6.0 to 8.0 and $25-55^{\circ}C$. The effect on the metal ions $Na^+$ and $K^+$ and (moderateactivity) $Mn^{2+}$ and $Ni^{2+}$ increased activity, while $Hg^{2+}$, $Cu^{2+}$, $Fe^{2+}$, and $Fe^{2+}$ inhibited activity. EDTA, phenylmethylsulfonyl fluoride and 5,5-dithiobis (2-nitrobenzoicacid) reduced activity, while tetrafluoroethylene and 1,10-phenanthroline inhibited activity. The amylase was highly tolerant of the surfactants TritonX-100, Tween-20, Tween-80 and compatible with organic solvents methanol, ethanol, isoamylalcohol, isopropanol, t-butylalcohol and the oxidizing agents hydrogen peroxide, sodium perborate and sodium hypochlorite, although potassium iodide and ammonium persulfate reduced activity. These properties suggest utility of pectinase and ${\beta}$-glucosidase produced by B. subtilis B22 under blue LED-mediated fermentation for industrial applications.

• 한국진공학회:학술대회논문집
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• 한국진공학회 2011년도 제40회 동계학술대회 초록집
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• pp.66-66
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• 2011

Anodic titanium dioxide (TiO2) nanotubes are very attractive materials for gas sensors due to its large surface to volume ratios. The most widely known method for fabrication of TiO2 nanotubes is anodic oxidation of metallic Ti foil. Since the remaining Ti substrate is a metallic conductor, TiO2 nanotube arrays on Ti are not appropriate for gas sensor applications. Detachment of the TiO2 nanotube arrays from the Ti Substrate or the formation of electrodes onto the TiO2 nanotube arrays have been used to demonstrate gas sensors based on TiO2 nanotubes. But the sensitivity was much lower than those of TiO2 gas sensors based on conventional TiO2 nanoparticle films. In this study, Ti thin films were deposited onto a SiO2/Si substrate by electron beam evaporation. Samples were anodized in ethylene glycol solution and ammonium fluoride (NH4F) with 0.1wt%, 0.2wt%, 0.3wt% and potentials ranging from 30 to 60V respectively. After anodization, the samples were annealed at $600^{\circ}C$ in air for 1 hours, leading to porous TiO2 films with TiO2 nanotubes. With changing temperature and CO concentration, gas sensor performance of the TiO2 nanotube gas sensors were measured, demonstrating the potential advantages of the porous TiO2 films for gas sensor applications. The details on the fabrication and gas sensing performance of TiO2 nanotube sensors will be presented.

• Fisheries and Aquatic Sciences
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• 제1권2호
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• pp.201-208
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• 1998

A protease, which had no tryptic and chymotryptic activity, was purified from the hepatopancreas of shrimp, P. japonicus, through ammonium sulfate fractionation, Q­Sepharose ionic exchange, benzamidine Sepharose 6B affinity, and Sephacryl S-100 gel chromatography. Molecular weight (M.W.) of the protease was estimated to be 24 kDa by gel filtration and showed a single peptide band by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protease had a low ratio of acidic to basic amino acids, which is different with pro teases from marine animals. The enzyme was partially inhibited by benzamidine, tosyl-L-lysine chioromethyl ketone (TLCK), phenylmethylsulfonyl fluoride (PMSF), soybean trypsin inhibitor (SBTI), and pepstatin. The enzyme did not have any activity against benzoyl-D,L-arginine p-nitroanilide (BAPNA) or benzoyl-L-tyrosine ethyl ester (BTEE) which is a specific substrate of trypsin and chymotrypsin, respectively. However, the enzyme showed activity forward N-CBZ-L-tyrosine p-nitrophenyl ester (CBZ-Tyr-pNE), N­CBZ-L-tryptophan p-nitrophenyl ester (CBZ-Trp-pNE), and N-CBZ-L-proline p-nitrophenyl ester (CBZ-Pro-pNE). The protease did not showed tryptic and chymotryptic activity, which was not reported in shrimp hepatopancreas.

• Journal of Microbiology and Biotechnology
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• 제6권1호
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• pp.1-6
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• 1996

A bacterial strain NS70 producing an alkaline protease was isolated from soil samples taken near a hot spring and identified as Bacillus licheniformis by its morphological and physiological properties and cellular fatty acid analysis. The isolated alkaline protease was purified by ammonium sulfate fractionation, DEAE-, CM-, and Phenyl-Sepharose column chromatography. The molecular weight of the purified enzyme was estimated to be 32, 000 Da by sodium dodecylsulfate polyacrylamide gel electrophoresis. Its optimal pH and temperature for proteolytic activity against Hammarsten casein were 12 and $65^{\circ}C$, respectively. The enzyme was stable at alkaline pH range from 6.0 to 12.0, and fairly stable up to $65^{\circ}C$. The enzyme was inhibited by phenylmethylsulfonyl fluoride but not by EDTA and N-ethylmaleimide indicating that the enzyme is serine protease. Enzyme activity was markedly inhibited by $Hg^{2+}$ and $Cu^{2+}$. Autolytic phenomena were observed on purified protease NS70 but autolysis was reduced by the addtion of $Ca^{2+}$ ion or bovine serum albumin.

• Membrane and Water Treatment
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• 제7권3호
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• pp.209-221
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• 2016

As a highly hydrophilic fibrillar mineral in nature, attapulgite (ATP) is a promising new additive for preparation of ultrafiltration (UF) hybrid membrane. In this work, ATP particles, which were grafted with a new Gemini surfactant of Ethyl Stearate-di(octadecyl dimethyl ammonium chloride) to detach the crystal bundles to single crystal and enhance the uniform dispersion in an organic polymer matrix, were incorporated into poly(vinylidene fluoride) (PVDF) matrix, and PVDF/Gemini-ATP hybrid membranes for adsorptive removal of Ni(II) ions from aqueous solution were prepared via a phase inversion method. Chemical composition, crystalization and morphology of the modified ATP were characterized by Fourier transform infrared spectroscopy (FTIR), Transmission electron microscope (TEM) and X-ray diffraction (XRD), respectively. The morphology of the hybrid membrane was characterized by Scanning electron microscopy (SEM), the performance of permeability, hydrophilicity and adsorption of Ni(II) ions were studied, and the adsorption kinetics of the PVDF/ATP hybrid membranes were particular concerned. The results showed that the hybrid membrane displayed a good thermal stability and hydrophilicity. Comparing with PVDF membrane, the hybrid membrane possessed good adsorption capacity for Ni(II) ions, and the adsorption kinetics fit well with Lagergren second-order equation.

• 한국물환경학회지
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• 제35권5호
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• pp.418-424
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• 2019

In this study, $TiO_2$ nanotubes with different morphologies were prepared in the electrolyte consisting of ethylene glycol, ammonium fluoride($NH_4F$), and deionized water($H_2O$) by controlling the voltage and time in the anodization method. Thicknesses and pore sizes of these $TiO_2$ nanotubes were measured to interpret the relationship between anodization conditions and $TiO_2$ nanotube morphologies. Element contents in the $TiO_2$ nanotubes were detected for further analysis of $TiO_2$ nanotube characteristics. Photoelectrolyticdecolorization efficiencies of the $TiO_2$ nanotube plates with various morphologies were tested to clarify the morphology that a highly active $TiO_2$ nanotube plate should have. Influences of applied voltage in photoelectrolysis processes and sodium sulfate($Na_2SO_4$) concentration in wastewater on the decolorization efficiency were also studied. To save the equipment investment cost in photoelectrolysis methods, a two-photoelectrode system that uses the $TiO_2$ nanotube plates as photoanode and photocathode instead of adding other counter electrodes was studied. Compared with single-photoelectrode system that uses the $TiO_2$ nanotube plate as photoanode and titanium plate as cathode on the view of the treatment of dye wastewater containing different amounts of salt. As a result, a considerably suitable voltage was strictly needed for enhancing the photoelectrolyticdecolorization effect of the two-photoelectrode system but if salts exist in wastewater, an excellent increase in the decolorization efficiency can be obtained.